The recent x-ray structure of mouse P-glycoprotein (Pgp) determined to 3.8 angstrom established the overall structural architecture of one of the most studied mammalian multidrug resistance (MDR) transporters. The structure of Pgp, however, is only a starting point for understanding its transport mechanism and potential inhibition. Clearly a higher resolution structure along with different conformations and co-crystal structures will be required to understand the detailed mechanism of transport coupled to ATP hydrolysis paving the way towards future rational drug design. We propose to (1) extend the resolution of Pgp crystals using very new membrane protein crystallization techniques and novel detergents to greatly improve the precision of the model, (2) determine additional conformations of Pgp both in the inward- and outward- facing conformations to map out the structural trajectories of the transport cycle, (3) determine the co-crystal structures of Pgp of two classical and clinically important compounds, doxorubicin and rhodamine, to understand poly-specific substrate binding, and (4) determine the x-ray structure of human Pgp or humanized version of Pgp, which will be very useful for drug design in the future. PUBLIC HEALTH RELEVANCE: P-glycoprotein plays a significant role in drug efficacy and multidrug resistance to several diseases, including cancer and HIV. This high-impact project will provide a detailed molecular structural basis underlying its transport mechanism. The structures of Pgp should greatly facilitate the development of new drugs to circumvent multidrug resistance in the future.